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- EMDB-1172: Conformational transition of initiation factor 2 from the GTP- to... -

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Basic information

Entry
Database: EMDB / ID: EMD-1172
TitleConformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome.
Map datacryo-EM map for T. thermophilus ribosomal complex with initiation factor IF2 in the GTP state
Sample
  • Sample: Ribosome complex with initiation factor 2 in GTP state
  • Complex: 70S
  • RNA: messenger RNA
  • RNA: formyl-methionyl-RNA
  • Protein or peptide: initiation factor 2
Function / homologyTranslation initiation factor IF-2, bacterial-like / translational initiation
Function and homology information
Biological speciesThermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 11.5 Å
AuthorsMyasnikov AG / Marzi S / Simonetti A / Giuliodori AM / Gualerzi CO / Yusupova G / Yusupov M / Klaholz BP
CitationJournal: Nat Struct Mol Biol / Year: 2005
Title: Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome.
Authors: Alexander G Myasnikov / Stefano Marzi / Angelita Simonetti / Anna Maria Giuliodori / Claudio O Gualerzi / Gulnara Yusupova / Marat Yusupov / Bruno P Klaholz /
Abstract: Initiation of protein synthesis is a universally conserved event that requires initiation factors IF1, IF2 and IF3 in prokaryotes. IF2 is a GTPase essential for binding initiator transfer RNA to the ...Initiation of protein synthesis is a universally conserved event that requires initiation factors IF1, IF2 and IF3 in prokaryotes. IF2 is a GTPase essential for binding initiator transfer RNA to the 30S ribosomal subunit and recruiting the 50S subunit into the 70S initiation complex. We present two cryo-EM structures of the assembled 70S initiation complex comprising mRNA, fMet-tRNA(fMet) and IF2 with either a non-hydrolyzable GTP analog or GDP. Transition from the GTP-bound to the GDP-bound state involves substantial conformational changes of IF2 and of the entire ribosome. In the GTP analog-bound state, IF2 interacts mostly with the 30S subunit and extends to the initiator tRNA in the peptidyl (P) site, whereas in the GDP-bound state IF2 steps back and adopts a 'ready-to-leave' conformation. Our data also provide insights into the molecular mechanism guiding release of IF1 and IF3.
History
DepositionSep 14, 2005-
Header (metadata) releaseOct 27, 2005-
Map releaseOct 27, 2006-
UpdateSep 19, 2012-
Current statusSep 19, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1172.gif

Downloads & links

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Map

FileDownload / File: emd_1172.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM map for T. thermophilus ribosomal complex with initiation factor IF2 in the GTP state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3 Å/pix.
x 120 pix.
= 360. Å		3 Å/pix.
x 120 pix.
= 360. Å		3 Å/pix.
x 120 pix.
= 360. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.333
CCP4 map header333
EM Navigator Movie #12.4192.4192.419
Density

Histogram

Histogram (log scale)
Contour Level1: 4.2 / Movie #1: 3.5
Minimum - Maximum-23.7895 - 30.7364
Average (Standard dev.)0.121324 (±2.44677)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 360 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z360.000360.000360.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-77-770
NX/NY/NZ15515578
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-23.78930.7360.121

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Supplemental data

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Sample components

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Entire : Ribosome complex with initiation factor 2 in GTP state

EntireName: Ribosome complex with initiation factor 2 in GTP state
Components
  • Sample: Ribosome complex with initiation factor 2 in GTP state
  • Complex: 70S
  • RNA: messenger RNA
  • RNA: formyl-methionyl-RNA
  • Protein or peptide: initiation factor 2

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Supramolecule #1000: Ribosome complex with initiation factor 2 in GTP state

SupramoleculeName: Ribosome complex with initiation factor 2 in GTP state
type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 4
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: 70S

SupramoleculeName: 70S / type: complex / ID: 1 / Name.synonym: 70S / Details: T.thermophilus; Thermus thermophilus 70S ribosome / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)

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Macromolecule #1: messenger RNA

MacromoleculeName: messenger RNA / type: rna / ID: 1 / Name.synonym: mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString:
GGCAAGGAGG UAAAAAUGAA AAAAAAA

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Macromolecule #2: formyl-methionyl-RNA

MacromoleculeName: formyl-methionyl-RNA / type: rna / ID: 2 / Name.synonym: fMetRNA / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString:
CGCGGGGGGA GCAGCCUGGU AGCUCGUCGG GCUCAUAACC CGAAGGUCGU CGGUUCAAAU CCGGCCCCCG CAACCA

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Macromolecule #3: initiation factor 2

MacromoleculeName: initiation factor 2 / type: protein_or_peptide / ID: 3 / Name.synonym: IF2 / Details: T.thermophilus; Factor with GTP analogue - GMPPCP / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Location in cell: cytoplasm
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET30b
SequenceGO: translational initiation
InterPro: Translation initiation factor IF-2, bacterial-like

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5
Details: 20mM Tris-HCl (pH7.5) ,20mM MgCl2, 100mM KCl, 1mM DTT
StainingType: NEGATIVE / Details: no staining, cryo-EM with holey carbon grids
GridDetails: 300 mesh Copper/Rhodium grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home-made cryo-plunger / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 77 K
Alignment procedureLegacy - Astigmatism: lens astigmatism was corrected at 50,000 times magnification
DateMar 11, 2004
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 3 µm / Number real images: 36 / Average electron dose: 10 e/Å2 / Details: Heidelberg Druckmaschinen / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: ide entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected using an semi-automatic selection procidure
CTF correctionDetails: individual particles
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: IMAGIC / Details: exact filtered back-projection / Number images used: 74300
Final angle assignmentDetails: beta gamma
Final two d classificationNumber classes: 2810

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Atomic model buiding 1

SoftwareName: program O
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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